In breast cancer, collagen re-alignment is predictive of subtype, outcome and recurrence with limited data on contribution to racial disparities. For African-American (AA) women, breast cancer mortality is higher than any other race/ethnicity, in spite of having lower incidence rates. A disproportionate number of African-American women are affected by aggressive metastatic triple negative breast cancer (TNBC) without significant knowledge of mechanisms driving this ancestry-dependent difference. Collagen processing is a primary feature of TNBC tumors with increases in tumor adjacent collagen deposition, altered tumor-stroma ratios, and re-alignment of collagen fibers at tumor borders leading to metastasis. However, a knowledge gap exists on understanding ancestry-dependent translational and post-translational mechanisms of the collagen structure in any breast cancer type progression. Our preliminary data reports, for the first time, that post-translational regulation of the collagen triple helical structure by hydroxylated prolines (HYP) is a defining mechanism of racial disparities in normal and TNBC tissue. Consequently, this proposal focuses on identifying ancestry-dependent translational and post-translational mechanisms of collagen re-alignment that can ultimately be used to predict and potentially prevent breast cancer metastasis in AA women. In Aim 1, we will define ancestry dependent post-translational HYP regulation of collagen sequences from low grade to invasive triple negative breast cancers. Novel technological platforms will be combined to measure collagen sequences from the tissue microenvironment with measurement of collagen fiber changes and immune infiltrate. In Aim 2, we will identify HYP post-translational regulation of collagen sequence variants in ancestry-mapped normal breast tissue stratified by density, socioeconomic status, marital status, contraceptive use, and risk factors of smoking and alcohol. We will further determine if collagen sequence variants can be used as a companion scoring metric for prediction of breast cancer risk. In Aim 3, ancestry-defined normal and metastatic human breast cells will be used to precisely trace ancestry dependent collagen processing and oncogenic signaling influenced by collagen sequence variants. This study will increase our understanding of racial disparities in metastatic collagen re-alignment, working to generate better ancestry-dependent biomarkers for earlier detection of breast cancer and limit mortality due to breast cancer in minority populations.